PROJECT SUMMARY Lyme disease is the leading tick-borne bacterial disease in the world resulting an estimated 300,000 cases per year in the US alone. Lyme disease is caused by tick-bite transmission of the pathogenic spirochete Borrelia burgdorferi (Bb). An increased understanding of the molecular mechanisms that Bb uses to survive throughout its infectious cycle is critical for the development of innovative diagnostic and therapeutic protocols to reduce the incidence of Lyme disease. Bb harbors a reduced genome lacking many canonical metabolic, virulence and host defense evasion functions and a large number of the genes in the Bb genome encode hypothetical proteins of unknown function uniquely conserved among Borrelia species, together suggesting that Bb has evolved unique mechanisms to survive in the host that remain largely unknown. In the previous funding cycle, we addressed one of the key challenges in the field of Bb molecular pathogenesis, definition of the transcriptional activity of Bb during mammalian infection. Our novel synergistic approach combined development and application of an in vivo expression technology (IVET)-method for Bb, genome-wide identification of the 5' end transcriptome of Bb during growth in culture and infectivity analysis of targeted Bb mutants. This work contributed significant insight into the Bb transcriptome in vitro and during infection and resulted in our discovery of a large number of novel infection-expressed genes likely to contribute to Bb pathogenesis. These findings catalyze our current efforts to address a critical unresolved gap in knowledge of the biology of this pathogen, which is elucidation of the molecular mechanisms that allow Bb to overcome barriers to infection in order disseminate to distal tissues, leading to persistent infection and therefore, the incapacitating symptoms of Lyme disease. The goal of this proposal is to elucidate the molecular mechanisms that Bb uses to overcome host barriers to infection in order to disseminate. To achieve this goal we have focused our studies on functional analysis of three novel genes, bb0318, bbk13 and bb0562, which our data indicate play critical roles in Bb infectivity and dissemination due to their distinct contributions to Bb's ability to evade the early immune responses of the host. We will elucidate the molecular mechanisms of bb0318-dependent resistance to oxidative stress and immune-cell killing (Aim 1) and bbk13-dependent evasion of host complement (Aim 2) as well as define the contribution of bb0562 to Bb infection (Aim 3). We propose a multidisciplinary approach for mechanistic analysis of each of these novel genes, using cutting edge technologies in biochemistry, cell biology, microscopy and innate immunity as well as in vivo live imaging, a diversity of mouse models and an experimental tick-mouse infectious cycle. The insight gained from our work will significantly impact the understanding of the mechanisms of Bb dissemination, which in turn will provide a foundation for novel approaches for Lyme disease interventions.